Isolated hearts were perfused in modified Krebs-Henseleit buffer (118 mM NaCl, 5.9 mM KCl, 1.07 mM free Ca2+, 0.5 mM free Mg2+, 25 mM NaHCO3, and 10 mM glucose, pH 7.4 at 38oC) containing 0, 1.2 or 5 mM Pi. The total H2O content was 0.853 ml/g wet wt. of which 59.3% (14C-mannitol space) was extracellular and independent of [Pi]. No significant change in intracellular Pi content was found with values ranging from 15.8 to 20.5 umol/ml cell H20. Increasing Pi from 0 to 5 mM significantly decreased total [Mg2+] from 17.0 to 16.2 umol/ml. From the citrate/isocitrate ratio, free intracellular [Mg2+] was 0.36, 0.44 and 0.04 umol/ml for 0, 1.2 and 5 mM Pi. The calculated intracellular pH from tissue HCO3- was 6.98, 7.19 and 7.15 respectively. As Pi was increased from 0 to 5 mM, the [CI-] decreased from 51.7 to 11.6 umol/ml. In contrast, intracellular [K+] was invariant at 128 to 132 umol/ml giving a membrane potential of about -83 mV. The intracellular [Na+] with increasing Pi was 1.62, 3.68 and 2.03 umol/ml. The cytosolic [ATP][ADP][Pi] from 3PGK-GAPDH-LDH equilibria decreased from 9,300 M-1 to 3,360 M-1 in the 0 mM and 5 mM Pi perfused hearts. The free energy of ATP hydrolysis was found to be -13.1, -13.3 and -13.2 kcal/mole for 0, 1.2 and 5 mM Pi hearts. The energy of the plasma membrane gradients of [Na+]o3[K+]i2[Cl-]o/[Na+]i3[K+]o2[Cl-]i were +12.7, +11.3 and +13.2 kcal/mole, or 97%, 85% and 99% of the cellular phosphorylation potential. It was concluded that the extent and direction of the major inorganic ions and membrane potential across constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis.